The cultivation of mammalian cells in a chemically defined protein-free medium has been known for more than 30 years (Evans, V. J., et al., Cancer Res. 16:77-86 (1956); Evans, V. J., et al., Am. J. Hyg. 70:297-302 (1959)); likewise, various cell lines, which have been established in protein-free medium (Evans, V. J., et al., Exp. Cell Res. 32:212-217 (1963); Cinatl and Vesely, Folio Biologica 13:61-67 (1967); Takoaka and Katsuta, Exp. Cell Res. 67:295-304 (1971); Sanford and Evans, J. Natl. Cancer Ins. 68:895-913 (1982); Okaba, T., et al., Proc. Natl. Acad. Sci. USA 81:453-455 (1984); Yamaguchi, N., et al., J. Natl. Cancer Inst. 75:29-35 (1985); Yamaguchi and Kawai, Cancer Res. 46:5353-5359, (1986); Kovar and Franek, Biotechnol. Lett. 9:259-264 (1987); Cinatl, J. Jr., et al., J. Biol. Stand. 16:249-257 (1988); Hill, M., et al., In Vitro Cell. Dev. Biol. 25:49-56 (1989); Rikimaru, K., et al., In Vitro Cell. Dev. Biol. 26:849-856 (1990)). Media which are available as commercial products and which enable cells to be grown in the absence of foreign proteins have only recently been developed (Fike, R., et al., In Vitro Cell. Dev. Biol. 26:54A (1990); Darfler, F. J., In Vitro Cell. Dev. Biol. 26:769-778 (1990)).
These known protein-free media are primarily suitable for growing continuous cell lines in a suspension, e.g., hybridomas (Kovar and Franek, Biotechnol. Lett. 9:259-264 (1987); Fike, R., et al., In Vitro Cell. Dev. Biol. 26:54A (1990); Darfler, F. J., In Vitro Cell. Dev. Biol. 26:769-778 (1990)), but on the other hand are frequently found to be unsuitable for promoting cell growth in monolayer cultures (Kovar, J., In Vitro Cell. Dev. Biol. 25:395-396 (1989)).
The significant influence of the properties of the culture surface on the growth of monolayer cultures in protein-free medium has been described (Sanford and Evans, J. Natl. Cancer Ins. 68:895-913 (1982); Price and Sanford, TCA Manual 2:379-382 (1976); Cinatl, J. Jr., et al., In Vitro Cell. Dev. Biol. 26:841-842 (1990)). Rappaport et al. found that the adhesion and growth of cells depended on a critical number of negative charges on the surface. They treated a glass flask by a special method with alkali which enabled the cultivation of various cell lines in a protein-free medium without a previous adaption phase (Rappaport, C., et al., Exp. Cell Res. 20:465-510 (1960)). This technique of preparing the culture surface is, however, complicated and restricted to glass. Besides, different artificial surfaces from various suppliers were tested in assays using cells in serum-containing media and other protein additives. The results of these investigations showed that for the growth of cells in protein-free media, a different quality of the culture-surface is required as for the growth of cells in serum-containing media.
It has been known for years that the properties of the surface of glass or plastics has a significant influence on the growth of monolayer cells in protein-free medium [Sanford and Evans, J. Natl. Cancer Ins. 68:895-913 (1982); Price and Sanford, TCA Manual 2:379-382 (1976); Cinatl, J. Jr., et al., In Vitro Cell. Dev. Biol. 26:841-842 (1990)). It can be seen from the literature that not only the composition but also the properties of the culture surface must be optimized in order to achieve a satisfactory growth of cells in such a medium.
Polyvinylformal (PVF) was used for the first time as culture surface by Barski and Maurin for various cell types in media containing serum, embryonal extract and Tyrodes' salt solution. Other authors used PVF for some time for growing various cell lines in media containing protein additives (Furusawa, K., et al., Kenkyu Hokoku-Sen'i Kobunshi Zairyo Kenkyosho 124:17-21 (1980); Craig, A. M., et al., J. Biol. Chem. 264:9682-9689 (1989); Nordby, A., et al., Invest. Radiol. 24:703-710 (1989)).
Commercial disposable plastics articles, including polystyrene (PS), possess mainly negative surface charges. Maroudas has established that a negative charge density of 2-10.times.10.sup.14 per cm.sup.2 is optimal for cell proliferation, e.g., PS-TC (polystyrene-tissue culture grade) has a negative charge density of 4-7.times.10.sup.14 per cm.sup.2. On the other hand, PVF has a negative charge density of only 0.4-0.8.times.10.sup.14 per cm.sup.2.